Chelating compounds

ABSTRACT

The invention provides novel condensed macrocyclic chelants of formula [X(CR 2  R 3 ) n  ] m , (wherein each X independently represents an oxygen or sulphur atom or a group of formula NH, N(CR 2  R 3 ) p  R 4 , N(CR 2  R 3 ) p  Y, or N(CR 2  R 3 ) n-  N((CR 2  R 3 ) p  Y) 2  ; each Y independently represents a group COZ, SO 2  Z, POZ 2 , CON(OH)R 2 , CH 2  SR 2 , CS 2  R 2  or CSZ; each Z independently represents a group OR 2  or NR 2  R 2  ; n is an integer of 1 to 4; m is an integer of 3 to 8; p is an integer of 1 to 3; each R 2  independently represents a hydrogen atom or a C 1-8  alkyl group optionally mono- or poly-substituted by hydroxyl or C 1-8  alkoxy groups and at least one pair of R 2  groups, in each pair one being from a (CR 2  R 3 ).sub. n moiety and the other being from a (CR 2  R 3 ) n  or (CR 2  R 3 ) p  moiety, together with the intervening atoms represents a 5 to 8 membered, saturated fused ring optionally containing one or more ring heteroatoms selected from nitrogen, oxygen and sulphur, said fused ring optionally being substituted by a group R 1  ; each R 1  independently represents a group R 4  or a nitrogen-attached group (CR 2  R 3 ) p  Y; each R 3  independently represents a hydrogen atom or a C 1-8  alkyl or C 1-8  alkoxy group optionally mono or poly substituted by hydroxy or C 1-8  alkoxy groups; and each R 4  independently represents a hydrogen atom, a halogen atom or a hydroxyl group or an optionally mono- or poly-hydroxylated C 1-8  alkyl, C 1-8  alkoxy, (C 1-8  alkoxy)-C 1-8  alkyl or poly-(C 1-8  alkoxy)-C 1-8  alkyl group, a sulphonate group or a group (CR 2  R 3 ) p  Y; with the proviso that at least 2 Y groups are present). These chelants are useful in the preparation of diagnostic and therapeutic agents, in particular chelate complexes suitable for use as contrast agents for diagnostic imaging techniques, e.g. MRI.

CHELATING COMPOUNDS

The present invention relates to certain novel chelating agents, inparticular polyamines, and to their uses, especially their medical uses.

The medical use of chelating agents is well established, for example asstabilizers for pharmaceutical preparations, as antidotes for poisonousheavy metal species and as diagnostic agents for the administration ofmetal species (e.g. ions or atoms) for diagnostic techniques such asX-ray, magnetic resonance imaging (MRI) or ultrasound imaging orscintigraphy.

Polyamine chelating agents, for example aminopoly-(carboxylic acid orcarboxylic acid derivative) (hereinafter APCA) chelating agents andtheir metal chelates, are well known and are described for example inU.S. Pat. No. 2,407,645 (Bersworth), U.S. Pat. No. 2,387,735(Bersworth), EP-A-71564 (Schering), EP-A-130934 (Schering), EP-A-165728(Nycomed AS), DE-A-2918842 (Rexolin Chemicals AB), DE-A-3401052(Schering), EP-A-258616 (Salutar), DE-A-3633245 (Schering), EP-A-263059(Schering), EP-A-277088 (Schering) and DE-A-3633243 (IDF).

Thus, for example, EP-A-71564 describes paramagnetic metal chelates, forwhich the chelating agents are nitrilotriacetic acid (NTA),N,N,N',N'-ethylenediamine-tetraacetic acid (EDTA),N-hydroxyethyl-N,N',N'-ethylenediamine-triacetic acid (HEDTA),N,N,N',N'',N''-diethylenetriamine-pentaacetic acid (DTPA) andN-hydroxyethylimino-diacetic acid, as being suitable as contrast agentsfor MRI, contrast being achieved by the effect of the magnetic field ofthe paramagnetic species (e.g. Gd(III)) with the chelating agentsserving to reduce the toxicity and to assist administration of thatparamagnetic species. Amongst the particular metal chelates disclosed byEP-A-71564 was Gd DTPA, the use of which as an MRI contrast agent hasrecently received much attention. The Gd(III) chelate of1,4,7,10-tetraazacyclododecanetetra-acetic acid (DOTA), referred to inDE-A-3401052 (Schering) and in U.S. Pat. No. 4,639,365 (University ofTexas), has also recently received attention in this regard.

To improve stability, water solubility and selectivity, relative to theAPCA chelating agents described in EP-A-71564, Schering, in EP-A-130934,have proposed the partial substitution for the N-attached carboxyalkylgroups of alkyl, alkoxyalkyl, alkoxycarbonylalkyl oralkylaminocarbonylalkyl groups, where any amide nitrogens may themselvescarry polyhydroxyalkyl groups. More recently, to improve compatibility,stability, solubility and selectivity, in EP-A-250358 Schering haveproposed a narrow range of compounds having a DTPA-like structureincluding a bridging alkylene chain. In the field of hepatobiliary MRIcontrast agents, where lipophilicity rather than hydrophilicity isdesired, Nycomed in EP-A-165728, have proposed the use of paramagneticchelates of certain anilide group-containing iminodiacetic acids andLauffer in WO-A-86/06605 has suggested the use of paramagnetic chelatesof triaza and tetraaza macrocycles which carry a fused aromatic ring butare otherwise unsubstituted.

Nycomed, in EP-A-299795, suggest that the toxicity of certain APCAchelating agents and their chelates may be reduced by introducing atleast one hydrophilic moiety as a substituent on one or more of thealkylene bridges between the amine nitrogens.

However, all hitherto known APCA chelating agents and their metalchelates encounter problems of toxicity, stability or selectivity andthere is thus a general and continuing need for such polyamine chelatingagents which form metal chelates of reduced toxicity, improved stabilityor improved water solubility.

We now propose a novel class of polyamine chelating agents whichincorporate within their structure a heterocyclic ring.

Thus viewed from one aspect the present invention provides a compound offormula I ##STR1## wherein each X independently represents an oxygen orsulphur atom or a group of formula NH, N(CR² R³)_(p) R⁴, N(CR² R³)_(p)Y, or N(CR² R³)_(n) --N((CR² R³)_(p) Y)₂ ;

each Y independently represents a group COZ, SO₂ Z, POZ₂, COH(OH)R², CH₂SR², CS₂ R² or CSZ;

each Z independently represents a group OR² or NR² R² ;

n is an integer of 1 to 4, preferably 2 or 3;

m is an integer of 3 to 8, preferably 3 to 6;

p is an integer of 1 to 3, preferably 1;

each R² independently represents a hydrogen atom or a C₁₋₈ alkyl groupoptionally mono- or poly-substituted by hydroxyl or C₁₋₈ alkoxy groupsand at least one pair of R² groups, in each pair one being from a (CR²R³)_(n) moiety and the other being from a (CR² R³)_(n) or (CR² R³)_(p)moiety (particularly preferably R² groups in (CR² R³)_(n) and (CR²R³)_(p) or (CR² R³)_(n) and (CR² R³)_(n) groups bonded to the same Xgroup), together with the intervening atoms represents a 5 to 8membered, preferably 5 or 6 membered, saturated fused ring optionallycontaining one or more, especially 1 or 2 ring heteroatoms selected fromnitrogen, oxygen and sulphur, said fused ring optionally beingsubstituted by a group R¹ ; each R¹ independently represents a group R⁴or a nitrogen-attached group (CR² R³ )_(p) Y;

each R³ independently represents a hydrogen atom or a C₁₋₈ alkyl or C₁₋₈alkoxy group optionally mono or poly substituted by hydroxy or C₁₋₈alkoxy groups; and each R⁴ independently represents a hydrogen atom, ahalogen atom or a hydroxyl group or an optionally mono-orpoly-hydroxylated C₁₋₈ alkyl, C₁₋₈ alkoxy, (C₁₋₈ alkoxy)-C₁₋₈ alkyl orpoly-(C₁₋₈ alkoxy)-C₁₋₈ alkyl group, a sulphonate group or a group (CR²R³)_(p) Y; with the proviso that at least 2 Y groups, preferably atleast 3, are present) or a chelate complex or salt thereof.

In the compounds of the invention, alkyl or alkylene moieties in groupsR¹ to R⁴, unless otherwise stated, may be straight chained or branchedand preferably contain from 1 to 8, especially preferably 1 to 6 andmost preferably 1 to 4, carbon atoms. Where substituents may themselvesoptionally be substituted by hydroxyl or alkoxy groups, this may bemonosubstitution or polysubstitution and, in the case ofpolysubstitution, alkoxy or hydroxyl substituents may be carried byalkoxy substituents.

Where, as is particularly preferred, the compounds of the inventionincorporate one or more hydrophilic R¹ to R⁴ groups, these arepreferably straight-chained or branched moieties having a carbon atomcontent of from 1 to 8, especially preferably 1 to 6, carbon atoms. Thehydrophilic groups may be alkoxy, polyalkoxy, hydroxyalkoxy,hydroxypolyalkoxy, polyhydroxyalkoxy, polyhydroxylated polyalkoxy,hydroxyalkyl, polyhydroxyalkyl, alkoxyalkyl, polyalkoxyalkyl,hydroxylated alkoxyalkyl, polyhydroxylated alkoxyalkyl, hydroxylatedpolyalkoxyalkyl, or polyhydroxylated polyalkoxyalkyl groups. Morepreferably however they will be monohydroxyalkyl or polyhydroxyalkylgroups. The hydrophilic groups serve to increase the hydrophilicity andreduce the lipophilicity of the metal chelates formed with the chelatingagents of the invention and it is preferred that the compounds offormula I should contain at least 1, conveniently from 1 to 4, andpreferably 1, 2 or 3 such hydrophilic groups. As hydrophilic groups,e.g. R¹ to R⁴ and (CR² R³)_(p) R⁴, the compounds of the invention maythus include for example hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl,1,2-dihydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl,2,3,4-trihydroxybutyl, 1-(hydroxymethyl)-2-hydroxy-ethyl, methoxymethyl,ethoxymethyl, 2-hydroxyethoxymethyl, methoxyethoxymethyl,(2-hydroxy-ethoxy)ethyl, etc, groups.

In the compounds of the invention, where two R² groups together with theintervening atoms form a fused cyclic group these fused groupspreferably are cyclopentane, cyclohexane, oxolane, oxane, thiolane,thiane, pyrrolidine, piperidine, piperazine, morpholine orperhydropyrimidine. (It will be appreciated that where the two R² groupsare not from the same (CR² R³)_(n) group the fused ring will beheterocyclic).

N-membered fused rings attached to the macrocyclic skeleton of themolecule at, or at positions adjacent a ring heteroatom of themacrocycle and rings attached at the 1 and 2 or 2 and N-1 positions ofthe fused ring are especially preferred. Moreover, in the compoundsaccording to the invention containing two or more fused rings, these arepreferably separated by at least one and especially at least two Xgroups. Particularly preferably, where there are two or more fusedrings, these will be spaced approximately uniformly about themacrocyclic skeleton.

It is also especially preferred that groups X comprising no ionizinggroup Y should be non-adjacent and particularly that they should adoptopposed positions in macrocyclic chelants, e.g. as the 1st and 3rd Xgroups in an 6 X ring. Particularly preferably such groups arehydrophilic groups such as are defined above.

Particularly preferred compounds of formula I include those of formulaIb ##STR2## where a and b are each zero, 1, 2 or 3, the sum of a and bbeing 1 to 3; d is 2,3 or 4;

n is as hereinbefore defined but preferably 2 or 3;

A is hydrogen or optionally hydroxylated, optionally alkoxylated alkylor CHR² Y;

R³ is hydrogen or optionally hydroxylated alkyl;

R² is hydrogen or optionally hydroxylated, optionally alkoxylated alkyland at least one pair of R² groups together with the intervening atomsform a fused ring of formula ##STR3## where G is a 3 to 7, preferably 3or 4, membered chain of carbon atoms and optionally a nitrogen, oxygenor sulphur atom, preferably a propylene or butylene chain, J is a 2 to5, preferably 2 or 3, membered chain of carbon atoms and optionally anitrogen, oxygen or sulphur atom, preferably an ethylene, propylene or2-oxa-propylene chain.

Especially prefered chelants for use according to the invention includethose of formulae Id to Ii ##STR4## where L is (CHR⁴)₂, (CHR⁴)₃ or CHR⁴OCHR⁴,

Q is (CHR⁴)₃ or (CHR⁴)₄,

u is 2 or 3,

and v is zero or 1.

Where a group NR₂ ² in a compound according to the invention is anitrogen attached heterocyclic ring, it will conveniently be of formula##STR5## where q is 0,1 or 2, and W is CHR⁴, NR⁴, O or S, where q iszero W preferably being CHR⁴. Particularly preferably such groups are offormula ##STR6##

In the compounds of formula I, the groups Y preferably representcarboxylic acid or amide groups, for example groups of formula COOH,CONH₂, CONCHR⁴ CHR⁴ W(CHR⁴)_(q) CHR⁴, CONHR^(2'') or CONR₂ ^(2'') (whereR^(2'') is an alkyl or mono or poly hydroxyalkyl group, for example aC₁₋₆ alkyl group optionally carrying 1, 2, 3 or 4 hydroxyl groups).

Where Y is a carboxyl group, the compounds of formula I can convenientlyform salts or chelates in which Y represents --COOM (wherein M⁺ is amonovalent cation or a fraction of a polyvalent cation, for example anammonium or substituted ammonium ion or a metal ion, for example analkali metal or alkaline earth metal ion). Particularly preferably, M⁺is a cation deriving from an organic base, for example meglumine orlysine. In such salts or chelates one or more (but not necessarily all)of the carboxyl groups are transformed into COOM groups.

It is particularly preferred that the number of the ion-forming groups Yin the compounds of formula I be chosen to equal the valency of themetal species to be chelated by the compound formula I. Thus, forexample, where Gd(III) is to be chelated, the compound of formula I (orsalt thereof) preferably contains three ion-forming Y groups, forexample --COOH (or --COOM). In this way, the metal chelate will beformed as a neutral species, a form preferred since the osmolalities inconcentrated solutions of such compounds are low and since theirtoxicities relative to their ionic analogues are significantly reduced.

Compounds of formula I in which all the Y groups are --COOH groups orsalts or amides of such compounds are especially preferred sincecompositions containing metal chelates of such compounds can readily besterilized, for example by autoclaving.

Included amongst the particularly preferred compounds according to theinvention are those of formulae Ib to Ii wherein each R² other thanthose forming fused rings represents a hydrogen atom or a mono- orpoly-hydroxylated alkyl group, Y represents a group of formula COZ and Zrepresents a hydroxyl group or a group NHR² and metal chelates and saltthereof.

Especially preferred compounds according to the invention include thoseof the following formulae Ij to Iaa ##STR7## preferably where all or allbut one group A is a CH₂ Y group and/or where at least one group A is anon-ionizing hydrophilic group, and where some or all of the R² and R⁴groups are hydrophilic groups, preferably for R² hydroxy- oralkoxy-alkyl groups and for R⁴ hydroxyl or hydroxy- or alkoxy- alkylgroups, especially where each A is hydrogen, hydroxypropyl, CH₂ COOH,CH₂ CON(CH₃)--CH₂ CHOHCH₂ OH, or CH₂ CONHR⁷ (where R⁷ represents CH₃,CH₂ CHOHCH₂ OH or CH(CH₂ OH)₂ or a group --CH₂ CONCH₂ CHR^(4'')W(CHR^(4''))_(q) CH₂ where W represents an oxygen atom or a group CH₂ orCHOH, q is 0 or 1 and R^(4'') is hydrogen or where q is 1 and W isoxygen each R^(4'') may also represent a C₁₋₄ hydroxyalkyl group) andthe metal chelates and the salts thereof.

Viewed from a further aspect, the invention also provides a process forthe preparation of the compounds of the invention, said processcomprising one or more of the following steps:

(a) reacting a compound of formula II

    [X.sup.' (CR.sup.2' R.sup.3').sub.n ].sub.m                (II)

(where R^(2') and R^(3') are as defined for R² and R³ or are protectedR² or R³ groups, and X' is a group X or a protected group X with theproviso that at least one X^(') group is of formula NH or (CR^(2')R^(3'))_(p) NH₂) with a compound of formula III

    Lv-(CR.sup.2' R.sup.3).sub.p -Y'                           (III)

(where Y' is a group Y or R⁴ or a protected group Y or R⁴, R^(2') R^(3')are as hereinbefore defined and Lv is a leaving group for example ahalogen atom, e.g. bromine or chlorine or a tosylate group and p is ashereinbefore defined) and if necessary subsequently removing anyprotecting groups used; and

(b) converting a compound of formula I into a chelate complex or saltthereof.

The compounds of formula II may be prepared in a number of ways usingtechniques known from the literature or analogous to literaturedescribed techniques. In particular, starting materials as described inBritish Patent Application No. 8900719.9 may be used.

Thus for example such compounds may be prepared by condensingbifunctional compounds of formula IV

    LvCO(CR.sup.2' R.sup.3').sub.n-1 [X.sup.'' (CR.sup.2' R.sup.3'.sub.n ].sub.j X.sup.'' (CR.sup.2' R.sup.3').sub.n-1 COLv        (IV)

(where R^(2') and R^(3') are as defined above, Lv is as defined above oris an alkoxy leaving group j is 0 to m-3, and mid-chain X'' groups, ifany, are groups X' and end of chain X'' groups are oxygen, sulphur orring nitrogen atoms or, preferably, NH groups) with a linking moleculeof formula V

    H[X''(CR.sup.2' R.sup.3').sub.n ].sub.i X''H               (V)

(where i is 1 to m-2 and R^(2'), R^(3') and X'' are as hereinbeforedefined) followed if necessary by removal of any protecting groups andif necessary by reduction.

The compounds of formula II may also be prepared by activating startingcompounds of formula V, e.g. by tosylation, and condensing the productwith a bifunctional compound of formula VI

    Lv(CR.sup.2' R.sup.3').sub.n [X.sup.'' (CR.sup.2' R.sup.3').sub.n ].sub.j+1 Lv                                                        (VI)

followed by removal of the tosyl and other protecting groups.

The compounds of formula II may also be prepared by cyclizing a linearcompound of formula VII

    PrNHCO(CR.sup.2' R.sup.3').sub.n-1 [X''(CR.sup.2' R.sup.3').sub.n ].sub.m-2 X''(CR.sup.2' R.sup.3').sub.n-1 COLv

(where R^(2'), R^(3') and X^('') are as defined above but where (CR^(2')R^(3')) moieties adjacent X^('') groups may represent carbonyl groups,Lv is a leaving group as defined above or is a hydroxyl group and Pr isa hydrogen atom or an amine protecting group) by reduction, tosylationand base-catalysed cyclization or by carboxyl activation cyclizationfollowed by reduction.

Where this procedure is followed, and where a polypeptide of formula VIIis used, selection of the amino acid precursors, e.g. use ofhydroxy-proline, enables a bicyclo compound of formula II to beproduced. Compounds of formula II may also be prepared by cyclization ofmacrocyclic compounds of formula VIII to produce the fused ring.

    [X.sup.'' (CR.sup.2' R.sup.3').sub.n ].sub.m               (VIII)

(where one X^('') is a NH group and one R^(2') is a group condensabletherewith, i.e. it carries a leaving group Lv, or where two R^(2')groups are condensable, e.g. one carries a leaving group and the othercarries a displacing group and the other groups, X'', R^(2') and R^(3')are as hereinbefore defined).

The compounds of formula II can also be prepared by reduction of cyclicimines of formula XIX

    [X''(CR.sup.2' R.sup.3').sub.n ].sub.m                     (XIX)

(wherein X'', R^(2') and R^(3') are as defined above but wherein a[X''(CR^(2') R^(3'))_(n) ]₂ X'' moiety represents an aromaticheterocyclic ring with two carbon attached --CR^(3') ═N-- groups)

Thus for example compounds of formula II can be prepared using thefollowing reaction schemes. ##STR8## This is similar to the procedure byTabushi et al. in Tetrahedron Letters 4339(1976) and 1049 (1977). Insome cases the use of metal ions such as Ni²⁺, K⁺ or Na⁺ as templates inthe cyclization step may catalyse the reaction or improve the yield.##STR9## This is similar to the traditional cyclization procedure ofRichmann et al. JACS 96:2268 (1974). ##STR10## t=1, 2 R²⁰ =R^(3') e.g.H, CH₃

The compounds of formula IX can be prepared from dicarbonyl derivativesin a Schiff's base condensation as described by Nelson in Pure andApplied Chemistry 52:461-476 (1980), e.g. ##STR11## R²¹ =H, R^(2'),R^(3') or an amino acid residue f=0 to 5 ##STR12## These are similar tothe procedure of Moi et al. JACS 110: 6266 (1988).

Where one R²¹ on the polypeptide of formula X results from use ofhydroxy proline, the resulting compound of formula (XI) carries a fusedgroup ##STR13## (where R²² is R^(2'), R^(3') or is a ring, e.g. a 5 or 6membered optionally hydrophilically substituted ring).

Reactions of this nature are described by Guerbet in EP-A-287465.##STR14## (where h is 3 or 4)

Further reaction schemes for the production of compounds of formula IIwill be evident to the skilled chemist from the literature, e.g. Tabushiet al. Tetr. Lett. 4339 (1976) and 1049 (1977), Richmann et al. JACS 96:2268 (1974), Nelson, pure and Applied Chemistry 52: 461-476 (1980). Moiet al. JACS 110: 6266 (1988), EP-A-287465 (Guerbet) Stetter et al.Tetrahedron 37: 767 (1981), EP- A-232751 (Squibb), Hancock et al. JACS110: 2788-2794 (1988), Smith et al. JACS 111: 7437-7443 (1989) and thereferences listed therein.

To introduce a (CR² R³)_(p) Y group onto a compound of formula II usingthe procedure of step (a) may be effected in an aqueous, preferablybasic medium, for example by using a halocarboxylic acid Hal (CR^(2')R^(3'))_(p) COOH or a metal, e.g. Li, salt thereof (where Hal is bromineor chlorine) followed by amidation or esterification of the carboxylgroup.

The introduction of (CR² R³)_(p) Y or (CR² R³)_(p) R⁴ moiety other thana carboxylic acid residue may for example be performed as follows:

a) To introduce a phosphonic acid moiety, the general method forsynthesis of alpha-aminophosphonic acids described by K. Moedritzer etal. in J. Org. Chem 31: 1603 (1966) may be used. ##STR15## (where R¹⁷NCH₂ Y is a compound of formula I). b) To introduce a hydroxamic acidmoiety, the general method for transformation of an activated acidderivative into hydroxamic acid described by P. N. Turowski et al. inInorg. Chem. 27: 474 (1988) may be used. ##STR16## (where R¹⁸ N(CH₂COOH)CH₂ Y is a compound of formula I). c) To introduce a sulfonic acidmoiety, synthesis may be performed by alkylation of an amino functionfor example with iodomethanesulfonic acid ##STR17## d) To introduce anonionizing (CR² R³)_(p) R⁴ group, synthesis may be performed byalkylation of an amino function with an optionally hydroxyl-protectedalkyl (or alkoxyalkyl, hydroxyalkyl etc) halide: ##STR18## followed ifnecessary by deprotection, e.g. debenzylation.

Amide derivatives of formula I may be produced from the oligo acids bymethods analogous to those of EP-A-250358 or of EP-A-299795. Furthermorehydrophilic substituents on the skeleton of the chelants of formula Imay be introduced by methods analogous to those of EP-A-299795.

Chelants of formula I may be used as the basis for bifunctional chelantsor for polychelant compounds, that is compounds containing severalindependant chelant groups, by substituting for one Y or R¹ to R⁴ groupa bond or linkage to a macromolecule or polymer, e.g. a tissue specificbiomolecule or a backbone polymer such as polylysine orpolyethyleneimine which may carry several chelant groups and may itselfbe attached to a macromolecule to produce a bifunctional-polychelant.Such macromolecular derivatives of the compounds of formula I and themetal chelates and salts thereof form a further aspect of the presentinvention.

The linkage of a compound of formula I to a macromolecule or backbonepolymer may be effected by any of the conventional methods such as thecarbodiimide method, the mixed anhydride procedure of Krejcarek et al.(see Biochemical and Biophysical Research Communications 77: 581(1977)), the cyclic anhydride method of Hnatowich et al. (see Science220: 613 (1983) and elsewhere), the backbone conjugation techniques ofMeares et al. (see Anal. Biochem. 142: 68 (1984) and elsewhere) andSchering (see EP-A-331616 for example) and by the use of linkermolecules as described for example by Nycomed in WO-A-89/06979.

Salt and chelate formation may be performed in a conventional manner.The chelating agents of the formula I (as defined above but with thedeletion of the second proviso) are particularly suitable for use indetoxification or in the formation of metal chelates, chelates which maybe used for example in or as contrast agents for in vivo or in vitromagnetic resonance (MR), X-ray or ultrasound diagnostics (e.g. MRimaging and MR spectroscopy), or scintigraphy or in or as therapeuticagents for radiotherapy, and such uses of these metal chelates form afurther aspect of the present invention.

Salts or chelate complexes of the compounds of the invention containinga heavy metal atom or ion are particularly useful in diagnostic imagingor therapy. Especially preferred are salts or complexes with metals ofatomic numbers 20-32, 42-44, 49 and 57 to 83, especially Gd, Dy and Yb.For use as an MR-diagnostics contrast agent, the chelated metal speciesis particularly suitably a paramagnetic species, the metal convenientlybeing a transition metal or a lanthanide, preferably having an atomicnumber of 21-29, 42, 44 or 57-71. Metal chelates in which the metalspecies is Eu, Gd, Dy, Ho, Cr, Mn or Fe are especially preferred andGd³⁺, Mn²⁺ and Dy³⁺ are particularly preferred. Chelates of ions ofthese metals specifically listed above with chelants of formula I(defined as above with the exclusion of the second proviso) or theirsalts with physiologically tolerable counterions are particularly usefulfor the diagnostic imaging procedures mentioned herein and they andtheir use are deemed to fall within the scope of the invention andreferences to chelates of compounds of formula I herein are consequentlyto be taken to include such chelates.

For use as contrast agents in MRI, the paramagnetic metal species isconveniently non-radioactive as radioactivity is a characteristic whichis neither required nor desirable for MR-diagnostics contrast agents.For use as X-ray or ultrasound contrast agents, the chelated metalspecies is preferably a heavy metal species, for example anon-radioactive metal with an atomic number greater than 37, preferablygreater than 50, e.g. Dy³⁺. For use in scintigraphy and radiotherapy,the chelated metal species must of course be radioactive and anyconventional complexable radioactive metal isotope, such as ^(99m) Tc or¹¹¹ In for example, may be used. For radiotherapy, the chelating agentmay be in the form of a metal chelate with for example ¹⁵³ Sm, ⁶⁷ Cu or⁹⁰ Y.

For use in detoxification of heavy metals, the chelating agent must bein salt form with a physiologically acceptable counterion, e.g. sodium,calcium, ammonium, zinc or meglumine, e.g. as the sodium salt of thechelate of the compound of formula I with zinc or calcium.

Where the metal chelate carries an overall charge, such as is the casewith the prior art Gd DTPA, it will conveniently be used in the form ofa salt with a physiologically acceptable counterion, for example anammonium, substituted ammonium, alkali metal or alkaline earth metal(e.g. calcium) cation or an anion deriving from an inorganic or organicacid. In this regard, meglumine salts are particularly preferred.

Viewed from a further aspect, the present invention provides adiagnostic or therapeutic agent comprising a metal chelate, whereof thechelating entity is the residue of a compound according to the presentinvention, together with at least one pharmaceutical or veterinarycarrier or excipient, or adapted for formulation therewith or forinclusion in a pharmaceutical formulation for human or veterinary use.

Viewed from another aspect, the present invention provides adetoxification agent comprising a chelating agent according to theinvention in the form of a weak complex or salt with a physiologicallyacceptable counterion, together with at least one pharmaceutical orveterinary carrier or excipient, or adapted for formulation therewith orfor inclusion in a pharmaceutical formulation for human or veterinaryuse.

The diagnostic and therapeutic agents of the present invention may beformulated with conventional pharmaceutical or veterinary formulationaids, for example stablizers, antioxidants, osmolality adjusting agents,buffers, pH adjusting agents, etc. and may be in a form suitable forparenteral or enteral administration, for example injection or infusionor administration directly into a body cavity having an external escapeduct, for example the gastrointestinal tract, the bladder or the uterus.Thus the agent of the present invention may be in a conventionalpharmaceutical administration form such as a tablet, capsule, powder,solution, suspension, dispersion, syrup, suppository, etc; however,solutions, suspensions and dispersions in physiologically acceptablecarrier media, for example water for injections, will generally bepreferred.

The compounds according to the invention may therefore be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art. For example, thecompounds, optionally with the addition of pharmaceutically acceptableexcipients, may be suspended or dissolved in an aqueous medium, with theresulting solution or suspension then being sterilized. Suitableadditives include, for example, physiologically biocompatible buffers(as for example, tromethamine hydrochloride), additions (e.g., 0.01 to10 mole percent) of chelants (such as, for example, DTPA, DTPA-bisamideor non-complexed chelants of formula I) or calcium chelate complexes (asfor example calcium DTPA, CaNaDTPA-bisamide, calcium salts or chelatesof chelants of formula I), or, optionally, additions (e.g., 1 to 50 molepercent) of calcium of sodium salts (for example, calcium chloride,calcium ascorbate, calcium gluconate or calcium lactate combined withmetal chelate complexes of chelants formula I and the like).

If the compounds are to be formulated in suspension form, e.g., in wateror physiological saline for oral administration, a small amount ofsoluble chelate may be mixed with one or more of the inactiveingredients traditionally present in oral solutions and/or surfactantsand/or aromatics for flavouring.

For MRI and for X-ray imaging of some portions of the body the mostpreferred mode for administering metal chelates as contrast agents isparentral, e.g., intravenous administration. Parenterally administrableforms, e.g., intravenous solutions, should be sterile and free fromphysiologically unacceptable agents, and should have low osmolality tominimize irritation of other adverse effects upon administration, andthus the contrast medium should preferably be isotonic or slightlyhypertonic. Suitable vehicles include aqueous vehicles customarily usedfor administering parenteral solutions such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection and other solutions suchas are described in Remington's Pharmaceutical Sciences, 15th ed.,Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and TheNational Formulary XIV, 14th ed. Washington: American PharmaceuticalAssociation (1975). The solutions can contain preservatives,antimicrobial agents, buffers and antioxidants conventionally used forparenteral solutions, excipients and other additives which arecompatible with the chelates and which will not interfere with themanufacture, storage or use of products.

Where the diagnostic or therapeutic agent comprises a chelate or salt ofa toxic metal species, e.g. a heavy metal ion, it may be desirable toinclude within the formulation a slight excess of the chelating agent,e.g. as discussed by Schering in DE-A-3640708, or more preferably aslight excess of the calcium salt of such a chelating agent. ForMR-diagnostic examination, the diagnostic agent of the presentinvention, if in solution, suspension or dispersion form, will generallycontain the metal chelate at concentration in the range 1 micromole to1.5 mole per liter, preferably 0.1 to 700 mM. The diagnostic agent mayhowever be supplied in a more concentrated form for dilution prior toadministration. The diagnostic agent of the invention may convenientlybe administered in amounts of from 10⁻³ to 3 mmol of the metal speciesper kilogram of body weight, e.g. about 1 mmol Dy/kg bodyweight.

For X-ray examination, the dose of the contrast agent should generallybe higher and for scintigraphic examination the dose should generally belower than for MR examination. For radiotherapy and detoxification,conventional dosages may be used.

Viewed from a further aspect, he present invention provides a method ofgenerating enhanced images of the human or non-human animal body, whichmethod comprises administering to said body a diagnostic agent accordingto the present invention and generating an X-ray, MR, ultrasound orscintigraphic image of at least a part of said body.

Viewed from a further aspect, the present invention provides a method ofradiotherapy practised on the human or non-human animal body, whichmethod comprises administering to said body a chelate of a radioactivemetal species with a chelating agent according to the invention.

Viewed from a further aspect, the present invention provides a method ofheavy metal detoxification practised on the human or non-human animalbody, which method comprises administering to said body a chelatingagent according to the invention in the form of its weak complex or saltwith a physiologically acceptable counterion.

Viewed from a yet further aspect, the present invention also providesthe use of the compounds, especially the metal chelates, according tothe invention for the manufacture of diagnostic or therapeutic agentsfor use in methods of image generation, detoxification or radiotherapypractised on the human or non-human animal body.

Viewed from a still further aspect, the present invention provides aprocess for the preparation of the metal chelates of the invention whichprocess comprises admixing in a solvent a compound of formula I or asalt (e.g. the sodium salt) or chelate thereof together with an at leastsparingly soluble compound of said metal, for example a chloride, oxide,acetate or carbonate.

Viewed from a yet still further aspect, the present invention provides aprocess for the preparation of the diagnostic or therapeutic agent ofthe present invention, which comprises admixing a metal chelateaccording to the invention, or a physiologically acceptable saltthereof, together with at least one pharmaceutical or veterinary carrieror excipient.

Viewed from a yet still further aspect, the present invention provides aprocess for the preparation of the detoxification agent of theinvention, which comprises admixing a chelating agent according to theinvention in the form of a salt with a physiologically acceptablecounterion together with at least one pharmaceutical or veterinarycarrier or excipient.

The disclosures of all of the documents mentioned herein areincorporated by reference.

The present invention will now be illustrated further by the followingnon-limiting Examples. All ratios and percentages given herein are byweight and all temperatures are in degrees Celsius unless otherwiseindicated.

EXAMPLE 113-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]N₄O₄ (morph)) ##STR19## Route (A)

a)4,8-Dioxo-13-oxa-3,6,9,15-tetraazabicyclo[9.3.1]-pentadecane([12]-dioxo-N.sub.4(morph))

Iminodiacetic acid diethylester (10 mmol) and2,6-bis-aminomethyl-morpholine (10 mmol) (described in British patentaplication no. 89 00719.9) are dissolved in ethanol and refluxed for 6days. After stripping to dryness, the resulting oil is chromatogrphed ona silica column.

b) 13-Oxa-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane ([12]N₄ (morph))

The product from a) is disolved in 1M borane in THF (15 equiv.) at 0° C.and stirred for 1 hour. The solution is refluxed overnight, cooled andexcess methanol is added. The reaction mixture is stripped and treatedwith a mixture of methanol and 6M HCl at ambient temperature. Afterevaporation of methanol, the pH of the solution is adjusted to 12 withNaOH, the reaction mixture is washed several times with chloroform andthe organic phase evaporated to dryness. Alternatively the alkalinewater phase is stripped to dryness, the residue dried by repeatedevaporation of ethanol and the title compound extracted from the residuewith dry ethanol. The resulting oil is purified on a silica column toyield the title compound.

c)13-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]N₄O₃ (morph))

The product from b) is treated with bromoacetic acid lithium salt (5equiv.) in water while the pH is kept between 9 and 11 with LiOH and thetemperature is gradually increased to 80° C. After reaction overnight,cooling and adjustment of pH to 3, the reaction mixture is treated witha strong cation exchanger. The product is separated from the resin bytreatment with saturated ammonia in water. The mixture is stripped todryness to yield the title compound The product is purified byprecipitation at pH 3-3.5 in water/alcohol, or by reversed phasechromatography.

Route (B) d)13-Oxa-3,6,9,15-tetrakis(tert.-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR20##

13-Oxa-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane (0.5 g; 2.3 mmol) wasdissolved in dry DMF. Potassium carbonate (1.6 g; 11.5 mmol) andtert-butyl bromoacetate (2.3 g; 11.5 mmol) were added and the reactionmixture was stirred at 50° C. overnight. The reaction mixture wascooled, evaporated to dryness, the residue was dissolved in chloroform(100 ml), washed with water (3×50 ml) and dried (MgSO₄). The chloroformwas removed and the crude title compound purified by flashchromatography (SiO₂ ; eluent chloroform:methanol 9:1). Yield: 0.5 g;32%. FAB MS: 671 (M+1).

f)13-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR21##

13-Oxa-3,6,9,15-tetrakis(tert.-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(0.4 g; 0.6 mmol) was dissolved in dichloromethane (3 ml) andtrifluoroacetic acid (3 ml) was added. The reaction mixture was stirredat ambient temperature overnight, concentrated and treated with diethyleither to give the title compound in quantitative yield. FAB MS: 447(M+1).

EXAMPLE 213-Hydroxy-4,8-bis(hydroxymethyl)-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]Dihydroxymethyl-N₄O₃ (hydroxypip)) ##STR22## Route (A)

a)2,10-Dioxo-4,8-bis(benzyloxymethyl)-13-hydroxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]Dibenzyloxymethyl-dioxo-N₄ (hydroxypip))

2,6-Bis(benzyloxymethyl)-1,4,7-triazaheptane (10 mmol), (described inWO-A-89/00557, Example 6 d) and2,6-bis(ethyloxycarbonyl)-4-hydroxy-piperidine (10 mmol) (described inWO-A-90/08138)is reacted as described in Example 1 a) to yield the titlecompound.

b)4,8-Bis(benzyloxymethyl)-13-hydroxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]Dibenzyloxymethyl-N₄(hydroxypip))

The product from a) is reduced with borane as described in Example 1 b)to yield the title compound.

c)4,8-Bis(hydroxymethyl)-13-hydroxy-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]Dihydroxymethyl-N₄ (hydroxypip))

The product from b) is dissolved in methanol and treated with 10%palladium on carbon at 50° C. overnight. After filtration andevaporation, the title compound is purified on a silica column.

d)13-Hydroxy-4,8-bis(hydroxymethyl)-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane([12]Dihydroxymethyl-N₄ O₃ (hydroxypip))

The product from c) is alkylated and purified as described in Example 1c) to yield the title compound.

Route (B)

The title compound is prepared using a scheme analogous to thatdescribed in Example 1, Route B.

EXAMPLE 37-Hydroxy-15-oxa-3,11,17,18-tetrakis(carboxymethyl)-3,11,17,18-tetraazatricyclo[11.3.1.1.⁵,9]octadecane ##STR23##

The title compound is prepared by the following reaction scheme:##STR24##

EXAMPLE 4 13-Oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane ##STR25##

a) 4-Benzyl-3,5-bis(tosylamidomethyl)morpholine ##STR26##

Tosyl chloride (5.5 g; 28 mmol) and triethylamine (2.9 g; 28 mmol) weredissolved in dry chloroform (100 ml) under nitrogen.4-Benzyl-3,5-bis(aminomethyl)-morpholine (prepared according toWO-A-90/08138) (6.8 g; 28 mmol), dissolved in dry chloroform (100 ml),was added dropwise during 1 hour. The reaction mixture was stirred atambient temperature overnight, washed with water (3×100 ml), dried(MgSO₄) and evaporated. The title compound was isolated afterchromatography on SiO₂, using ethyl acetate as eluent. Yield: 10.6 g(70%). FAB MS: 544 (M+1).

b)15-Benzyl-13-oxa-3,6,9-tris(tosyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR27##

4 Benzyl-3,5-bis(tosylamidomethyl)-morpholine (2.3 g; 4.2 mmol) wasdissolved in dry DMF (50 ml). Sodium hydride (50% in mineral oil, 1.1 g;8.4 mmol) was added and the temperature raised to 100° C. after 30 min.O,N,O'-Tris(tosyl)-diethanolamine (made according to the proceduredescribed in Can. J. Chemistry 45: 1555 (1967)) (2.4 g; 4.2 mmol)dissolved in dry DMF (100 ml), was added dropwise during 1 hour. Thereaction mixture was stirred at 100° C. overnight, cooled on ice/waterand 200 ml water was added dropwise. The precipitate was filtered offand dried. Yield: 8 g; 50%. FAB MS: 768 (M+1).

c) 13-Oxa-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane ##STR28##

15Benzyl-13-oxa-3,6,9-tris(tosyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(2 g; 2.6 mmol) was dissolved in concentrated sulfuric acid (10 ml) andheated to 100° C. for 48 hours. The reaction mixture was cooled onice/water and diethyl ether (30 ml) was added dropwise. The precipitatedpolyhydrosulfate salt was filtered under nitrogen, washed with smallvolumes of dry diethyl ether and dissolved in 50% sodium hydroxidesolution. The resulting mixture was extracted continuously withchloroform for 48 hours. The chloroform was removed to yield the titlecompound. Yield: 0.23 g, 42%. FAB MS: 215 (M+1).

d)13-Oxa-3,6,9-tris(tert.-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR29## 13-Oxa-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane (0.3 g: 1.4mmol) was dissolved in dry DMF. Sodium hydrogen carbonate (0.6 g; 7mmol) and tert-butyl bromoacetate (1.4 g; 7 mmol) were added and thereaction mixture was stirred at ambient temperature overnight. Thereaction mixture was evaporated to dryness, the residue was dissolved inchloroform (100 ml), washed with water (3×50 ml) and dried (MgSO₄). Thechloroform was removed and the crude title compound purified by flashchromatography (SiO₂ ; eluent chloroform:methanol 9:1). Yield: 0.47 g;60%. FAB MS: 557 (M+1).

e)13-Oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR30##

13-Oxa-3,6,9-tris(tert.-butoxycarbonylmethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(0.4 g; 0.7 mmol) was dissolved in dichloromethane (3 ml) andtrifluoroacetic acid (3 ml) was added. The reaction mixture was stirredat ambient temperature overnight, concentrated and treated with diethylether to give the title compound in quantitative yield. FAB MS: 389(M+1).

EXAMPLE 5 Gadolinium chelate of13-oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane

13-Oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(Example 4) (0.23 g; 0.6 mmol) was dissolved in water (2 ml) and the pHadjusted to 5 with 1M NaOH. Gadolinium oxide (0.11 g; 0.3 mmol) wasadded and the resulting mixture stirred at 90° C. for 24 hours. Thereaction mixture was filtered and the filtrate evaporated to dryness toyield 0.3 g (92%) of a yellow solid. FAB MS: 543 (M+1).

EXAMPLE 615-(2'-Hydroxypropyl)-13-oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane##STR31##

13-Oxa-3,6,9-tris(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(Example 4) (0.23 g; 0.6 mmol) is dissolved in water (2 ml) and the pHadjusted to 12 with 1M NaOH. The solution is cooled to ambienttemperature, and propylene oxide (0.05 g; 0.9 mmol) is added. Thereaction flask is stoppered, and left to stir at ambient temperatureovernight. The reaction mixture is acidified and treated with a cationexchange resin to give the title compound.

EXAMPLE 7 Sodium salt of the cadolinium chelate of13-oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo9.3.1]pentadecane

13-Oxa-3,6,9,15-tetrakis(carboxymethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadecane(Example 1) (0.22 g; 0.5 mmol) was dissolved in water (2 ml) and the pHadjusted to 5 with 1M NaOH. Gadolinium oxide (0.09 g; 0.25 mmol) wasadded and the resulting mixture stirred at 90° C. for 24 hours. Thereaction mixture was filtered and the filtrate evaporated to dryness toyield 0.3 g (92%) of a yellow solid. FAB MS: 622 (M+Na).

We claim:
 1. A compound of formula Ik ##STR32## wherein three groups Aare independently groups of formula CH₂ Y and the remaining A grouprepresents a hydrogen atom, a C₁₋₃ alkyl group optionally substituted bya group Y or, in the 15-position, a group R⁴ ;each Y independentlyrepresents a group --COZ, --POZ₂ or --CON(OH)R² ; each Z independentlyrepresents a group --OR² or --NR² R² ; each R² independently representsa hydrogen atom or a C₁₋₈ alkyl group optionally mono- orpoly-substituted or a pair of R² groups represent a bridging group whichtogether with the intervening two carbon and one nitrogen atoms form a 5to 8 membered saturated fused ring containing one nitrogen heteroatomand, where the ring is 6-membered, optionally one further ringheteroatom selected from nitrogen, oxygen and sulphur, said ringoptionally being substituted by a group R⁴ ; and each R⁴ independentlyrepresents a hydroxy group or an optionally mono- or poly-hydroxylatedand/or alkoxylated C₁₋₈ alkyl group; or a chelate complex or saltthereof.
 2. A compound as claimed in claim 1 being a compound of formulaIk wherein each R² is hydrogen and the A groups at the 3-, 6- and9-positions are carboxymethyl groups, or a chelate complex or saltthereof.
 3. A compound as claimed in claim 1 being a chelate complex ofa compound of formula Ik with a paramagnetic metal or other heavy metalion, or a salt thereof.
 4. A compound as claimed in claim 2 being achelate complex of a compound of formula Ik with a paramagnetic metal orother heavy metal ion, or a salt thereof.
 5. A compound as claimed inclaim 3 wherein said metal is selected from Eu, Gd, Dy, Ho, Cr, Mn andFe.
 6. A compound as claimed in claim 4 wherein said metal is selectedfrom Eu, Gd, Dy, Ho, Cr, Mn and Fe.
 7. A diagnostic or therapeuticcomposition comprising a metal chelate, whereof the chelating moiety isthe residue of a compound of formula Ik as defined in claim 1, togetherwith at least one pharmaceutical or veterinary carrier or excipient. 8.A method of generating enhanced images of the human or non-human animalbody, which method comprises administering to said body a diagnosticagent as claimed in claim 7 and generating an X-ray, MR, ultrasound orscintigraphic image of at least a part of said body.
 9. A compound offormula 1k ##STR33## wherein three groups A are independently groups offormula CH₂ Y and the remaining A group represents a hydrogen atom, aC₁₋₃ alkyl group optionally substituted by a group Y or, in the15-position, a group R⁴ ;each Y independently represents a group --COZ,--POZ₂ or --CON(OH)R² ; each Z independently represents a group --OR² or--NR² R² ; each R² independently represents a hydrogen atom or a C₁₋₈alkyl group optionally mono- or poly-substituted or a pair of R² groupsrepresent a bridging group which together with the intervening twocarbon and one nitrogen atoms form a 5 to 8 membered saturated fusedring containing one nitrogen heteroatom and, where the ring is6-membered, optionally one further ring heteroatom selected fromnitrogen, oxygen and sulphur, said ring optionally being substituted bya group R⁴ ; and each R⁴ independently represents a hydroxy group or anoptionally mono- or poly-hydroxylated and/or alkoxylated C₁₋₈ alkylgroup.